PROJECT SUMMARY/ABSTRACT The goal of this project is to decipher the functional role of testicular macrophages and other leukocytes in the pubertal testis after exposure to the Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP). Although these cells have traditionally been known for their phagocytic and antigen presentation functions, there is a growing body of evidence that these cells are critical for normal development, homeostasis and repair/regeneration of tissues after toxicant injury. We have previously described a robust infiltration of CD11b+ immunoreactive cells, representing macrophages, neutrophils, monocytes, natural killer and dendritic cells, into the testicular interstitial space in pubertal aged rats (PND 28) after MEHP (700 mg/kg, p.o.) treatment. The most pronounced infiltration of these cells occurs in pubertal rats, with lesser amounts in adult rats. Further, C57BL/6J mice at both ages do not have a significant infiltration due to MEHP exposure. The species and age-dependent sensitivity of MEHP- induced testicular injury is well recognized, although the mechanisms that account for these differences remain unresolved. A detailed evaluation of the cells that lie adjacent to the basement membrane of the seminiferous tubules revealed a specific testicular macrophage sub-population, the peritubular macrophages (ptM?s), that is increased in number and in specific regional localization along the periphery of the seminiferous tubules for a sustained period (>2 weeks) after MEHP exposure. The ptM? subtype has gained attention in the field of testis biology because they are predicted to play a critical role in the maintenance of the microenvironment supporting the spermatogonial niche. These findings have led to the hypothesis that MEHP-induced Sertoli cell injury incites an increase in the number and localization of distinct testicular macrophage subtypes as a mechanism to facilitate the efficient repair and recovery of spermatogenesis. To test this hypothesis, the first specific aim will characterize and determine the localization of macrophages subtypes in the pubertal testis in response to a range of MEHP doses (from a low of 10 mg/kg with increased dose intervals of 100, 250, 500 or 700 mg/kg). In the second aim, the cellular mechanisms that induce an infiltration of leukocytes into the testis will be defined. In the final aim, the extent that testicular macrophage subtypes are involved in mediating the recovery of spermatogenesis after a sub-chronic low dose MEHP treatment regimen will be determined as a strategy to more accurately translate the findings of this research project to human relevant exposures. Insights gained from this work will be valuable for predicting individuals susceptible to toxicant-induced infertility and the prevention of reproductive health risks associated with this ubiquitous environmental toxicant. Assessing the individual susceptibility to interactions of environmental toxicants with the immune system is consistent with goals of the NIEHS 2018-2023 strategic plan.